Animal Age Affects the Gut Microbiota and Immune System in Captive Koalas (Phascolarctos cinereus)

ABSTRACT Gut microbiota is one of the major elements in the control of host health. However, the composition of gut microbiota in koalas has rarely been investigated. Here, we performed 16S rRNA gene sequencing to determine the individual and environmental determinants of gut microbiota diversity and function in 35 fecal samples collected from captive koalas. Meanwhile, blood immune-related cytokine levels were examined by quantitative reverse transcription-PCR to initially explore the relationship between the gut microbiota and the immune system in koalas. The relative abundance of many bacteria, such as Lonepinella koalarum, varies at different ages in koalas and decreases with age. Conversely, Ruminococcus flavefaciens increases with age. Moreover, bacterial pathways involved in lipid metabolism, the biosynthesis of other secondary metabolites, and infectious disease show a significant correlation with age. Age affects the relationship between the microbiota and the host immune system. Among them, the gut microbiota of subadult and aged koalas was closely correlated with CD8β and CD4, whereas adult koalas were correlated with CLEC4E. We also found that sex, reproductive status, and living environment have little impact on the koala gut microbiota and immune system. These results shed suggest age is a key factor affecting gut microbiota and immunity in captive koalas and thus provide new insight into its role in host development and the host immune system. IMPORTANCE Although we have a preliminary understanding of the gut microbiota of koalas, we lack insight into which factors potentially impact captive koalas. This study creates the largest koala gut microbiota data set in China to date and describes several factors that may affect gut microbiota and the immune system in captive koalas, highlighting that age may be a key factor affecting captive koalas. Moreover, this study is the first to characterize the correlation between gut microbiota and cytokines in koalas. Better treatment strategies for infectious disorders may be possible if we can better understand the interactions between the immune system and the microbiota.

Q3.Line 51, "several lignin ... in the gastrointestinal tract of koalas." Which bacteria have been identified? Q4.The introduction is not detailed enough. In the third paragraph, you talked about immune response and cytokine. The introduction of koala cytokine and its previous study is not explicit and needs to be supplemented. Q5.Where is the sex-related cytokines conclusion? Q6.Line 65, "Gut microbiota can also help the host detoxify secondary compounds from plants." Please give us some examples. Q7.Line 24, "gut microbial" should be "gut microbiota". Q8.Line 38, "gut flora" should be "gut microbiota". Q9.Line 83, "the gut microbiota ...an endangered animal." The language is not professional. Should be: the research in gut microbiota .... Q10.Line 106-111, table 3 "N" should be in italic. Q11.Line 264, "gut mycobiome" spelling mistake. Q12.Line 381, Define each abbreviation and introduce it in parentheses the first time it is used. e.g., "C-Type Lectin Domain Family 4 Member(CLEC4E)" Q13.The figures are not big and clear enough. All the figures need to be improved. Q14. Figure 1,2 needs to be marked A, B. Q15.Use the italic system with the scientific name of gut microbiota in the figure legend. Q16. Figure 5 demonstrated diversity analysis, So the legend title should be described as diversity differences.
Reviewer #2 (Comments for the Author): This paper mainly describes the gut microbiota composition and the blood cytokine in different age captive koalas living in China. The author also analyzed whether sex, reproductive state, and living environment affected the captive koala. This study shows that individual factors are key factors in the growth and health of captive koalas. It is an interesting and original work. The reader will be interested in reading. However, there are some deficiencies that need to be corrected.
Q1.The abstract is incomplete. I cannot find the main conclusion in the abstract. Q2.Does not completely describe the main points of your research in the importance part. The author need to addition describe the significance and the innovation points of the study. Q3.The authors described the importance of studying gut microbiota. But didn't describe the previous study about koala gut microbiota. Similarly, the authors just talk about the cytokines being suitable targets for assessing immune responses. Please supplement the previous study about koalas. Q4. The number of ASVs mean SD = 768.54{plus minus}132.94. And the sample total of 35 in this study. Please check if the total number of ASVs is correct. Q5.The format of the Latin name is incorrect. All the scientific names of gut microbiota should in italic. "β" in the text and "P" in figure legends, etc should in italic. Q6.Many English mistakes were found in this manuscript, eg. "gut flora", and "gut mycobiome" should be "gut microbiota". Q7.Line 298, How many live in noisy koalas or quiet habitats koalas respectively? Q8.Line 215, the initial letters need to be capitalized. Q9.The resolution of the figures should be improved.

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Thank you for submitting your paper to Microbiology Spectrum. This paper mainly describes the gut microbiota composition and the blood cytokine in different age captive koalas living in China. The author also analyzed whether sex, reproductive state, and living environment affected the captive koala. This study shows that individual factors are key factors in the growth and health of captive koalas. It is an interesting and original work. The reader will be interested in reading. However, there are some deficiencies that need to be corrected.
Q1. The abstract is incomplete. I cannot find the main conclusion in the abstract.
Q2. Does not completely describe the main points of your research in the importance part.
The author need to addition describe the significance and the innovation points of the study.
Q3. The authors described the importance of studying gut microbiota. But didn't describe the previous study about koala gut microbiota. Similarly, the authors just talk about the cytokines being suitable targets for assessing immune responses. Please supplement the previous study about koalas.
Q4. The number of ASVs mean SD = 768.54±132.94. And the sample total of 35 in this study. Please check if the total number of ASVs is correct.
Q5. The format of the Latin name is incorrect. All the scientific names of gut microbiota should in italic. "β" in the text and "P" in figure legends, etc should in italic.
Q7. Line 298, How many live in noisy koalas or quiet habitats koalas respectively?
Answer and Revision: Thank you for your suggestion. "gut flora" was revised to "gut microbiota". Q9. Line 83, "the gut microbiota ...an endangered animal." The language is not professional. Should be: the research in gut microbiota .... Answer and Revision: Thank you for your suggestion. We have revised this sentence as "the research in gut microbiota and blood cytokine levels provides more specific insights into the koala's unique biology without harming or disturbing an endangered animal." Q10. Line 106-111, table 3 "N" should be in italic.
Answer and Revision: Thank you for your suggestion. "N" in table 3 was revised in italic.
Answer and Revision: Thank you for your suggestion. "gut mycobiome" was revised to "gut microbiota"  Q16. Figure 5 demonstrated diversity analysis, So the legend title should be described as diversity differences.
Answer and Revision: Thank you for your suggestion. We have revised the legend title in Figure 5 as "Diversity differences in the gut microbiota among the three age groups." Reviewer #2 (Comments for the Author): This paper mainly describes the gut microbiota composition and the blood cytokine in different age captive koalas living in China. The author also analyzed whether sex, reproductive state, and living environment affected the captive koala. This study shows that individual factors are key factors in the growth and health of captive koalas. It is an interesting and original work. The reader will be interested in reading.
However, there are some deficiencies that need to be corrected.
Q1. The abstract is incomplete. I cannot find the main conclusion in the abstract.
Answer and Revision: Thank you for your suggestion. We have added the conclusion to the abstract.
Q2. Does not completely describe the main points of your research in the importance part. The author need to addition describe the significance and the innovation points of the study.
Answer and Revision: Thank you for your suggestion. The significance and the innovative points of this study are to create the largest koala gut microbiota dataset in China to date and description several factors that may affect captive koalas on the gut microbiota and immune system, highlighting that age may be the key factor affecting captive koalas. Moreover, this study is the first to characterize the correlation between gut microbiota and cytokines in koalas. Better treatment strategies for infectious disorders may be possible if we can better understand the interactions between the immune system and the microbiome. We have added a description of this (confer Lines 40-46 of the revised manuscript).
Q3. The authors described the importance of studying gut microbiota. But didn't describe the previous study about koala gut microbiota. Similarly, the authors just talk about the cytokines being suitable targets for assessing immune responses. Please supplement the previous study about koalas.
Answer and Revision: Thank you for your suggestion. The previous study about koala gut microbiota is described in the first paragraph of the introduction part. In addition, we follow the suggestion that supplemented the introduction of koala cytokine and its previous study (confer Lines 76-79 of the revised manuscript).
Q4. The number of ASVs mean SD = 768.54{plus minus}132.94. And the sample total of 35 in this study. Please check if the total number of ASVs is correct.
Answer and Revision: Thank you for your suggestion. We have carefully checked and made sure the total number of ASVs (after rarefaction) is correct.
Q5. The format of the Latin name is incorrect. All the scientific names of gut microbiota should in italic. "β" in the text and "P" in figure legends, etc should in italic.
Answer and Revision: Thank you for your suggestion. We have carefully checked and revised the whole manuscript and revised the "β" and "P" as italic.
Answer and Revision: Thank you for your suggestion. We have revised "gut flora" and "gut mycobiome" to "gut microbiota".
Q7. Line 298, How many live in noisy koalas or quiet habitats koalas respectively? Answer and Revision: Thank you for your suggestion. The number of koalas living in noisy is 19 and in quiet habitats is 10). We have added a description of this in the text.
Q8. Line 215, the initial letters need to be capitalized.
Answer and Revision: Thank you for your suggestion. We have revised to capitalize the initial letters in this sentence.
Q9. The resolution of the figures should be improved.
Answer and Revision: Thank you for your suggestion. We have revised all the figures. Re: Spectrum04101-22R1 (Age was a key effect on the gut microbiota and immune system in captive koalas (Phascolarctos cinereus)) Dear Prof. Shining Guo: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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